Analysis of cell viability, especially the one of pollen grains, plays an important role at various aspects of plant breeding and plant production processes via seeds.
Analysis of the viability of unripe pollen grains (microspores) is important for the production of homozygous parental lines and for the development of the specific protocols or methods thereof. The production of parental lines from pollen grains safes numerous generations in the breeding cycle and is therefore considered as essential for efficient parental line production. However, for an optimal production of these lines their donor plants have to be grown under optimal conditions.
The analysis of pollen quality plays an important role during the selection of male-sterile mother lines. The use of male-sterile maternal lines, based on cytoplasmic male sterility, which is only inherited by the female, are preferred not only in insect and wind pollinated species but also in hand-pollinated ones. Because of the missing pollen, male sterile maternal lines are unable to self-pollinate, which prevents the contamination of F1 seeds with maternal material, and have the advantage that labour-intensive emasculation steps can be omitted.
High quality pollen is essential for an efficient F1 hybrid seed production. High pollen quality will give maximum seed set whereas lower qualities will result in a lower yield and therefore lead to higher production costs per seed. Pollen quality is affected by its genetic constitutions, growth conditions of the plants but also by pesticides. It is common practise to apply pesticides before and during production processes to keep the fruit-bearing plants healthy as it can take to up to 2 months before fruits and seeds, respectively, can be harvested. Therefore, the viability of pollen should be monitored continuously during seed production, to indicate/predict and control the production, to estimate the optimal time point of pollination, e.g. after pesticide application or to test, which pesticides are not suitable, or to check the effect of other chemicals (e.g. fertilizers), or growth conditions, respectively.
Another common practise in breeding and F1 hybrid seed production as well as in germplasm preservation programs is to work with stored pollen. For any storage program the highest quality pollen should be used. Routine pollen viability analysis in this field will allow the optimization of storage protocols and subsequent processes.
Currently, no efficient, high-throughput and easily applicable technique exists to analyse pollen quality on-site.
Pollen viability is generally determined by various classical methods like staining techniques or in vitro germination assays. However, the results from various staining techniques do not always correlate with the in vitro germination and it cannot be applied to all species. The staining techniques are based on enzymatic reactions that might not be present in the pollen of all plant species, and in vitro pollen germination depends highly on the correct conditions, which need to be adjusted individually. Both, the current methods analysing pollen viability and germination are limited in the number of cells that can be analysed in a certain time frame and they are time consuming in preparation and analysis.
Furthermore, the current techniques require next to specific chemicals various specific equipment like fluorescent microscopes, incubators, and a person skilled in the art. In general, this means that these analyses are performed in analytical laboratories and cannot be performed “on-site” between the plants in a greenhouse or open field. Finally, since these analyses are quite complex to perform and involve several error-prone steps from sample preparation to analysis, their reproducibility can vary tremendously at different locations spanning the whole seed production process, i.e. the different sites where these data are urgently needed. Thus, experience shows that pollen viability data obtained at different geographical locations and even in closely related analytical laboratories are not always comparable and suffer from subjective errors.
The need of reliable apparatuses and methods for evaluating the quality of pollen and plants is widely recognised. WO 2012/129199 A2 discloses such methods for the discrimination and sorting of pollen and the determination of pollen viability. For these analyses either conventional fluorescence-based flow cytometers or optical density measurements are required. For fluorescence-based analyses the presence/absence of genetic markers are mandatory in order to permit discrimination when specific dyes bind to the respective cells. Even though this technique provides statistical data, it is not suitable for on-site analyses and works only if such genetic markers are available. The optical density measurements, on the other hand, provides a means for discriminating lysing pollen, but could deliver confusing data if pollen cells are in different developmental stages with potentially varying optical densities but still viable characteristics. In this regard, WO 2012/129199 A2 refrains from disclosing supporting data.
An “on-site” analysis of e.g. pollen cells that are going to be used in F1 hybrid seed production is of huge commercial interest with respect to saving production and subsequent costs. In the case of low quality (low viability) pollen it can be decided not to use it for pollination, reducing the subsequent efforts of pollen storage procedures, mass hand pollinations, growing the material until fruit harvest, seed extraction and further processing. An “on-site” analysis of pollen of breeding material would allow the easy and direct selection on male-fertile/male sterile, heat- and/or salt-tolerant plants making the selection process more efficient as well as reducing the labour of growing and maintaining the selected individual plants.
Not only seed production, but also fruit production relies on fertilization of the flowers with good quality pollen. The unknown effects of pesticides on the one hand, as well as the need of mass hand pollinations on the other hand (for example because of the extinction the respective insects) are calling for a simple tool to determine the pollen quality.